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Methods Mol Biol. 2017;1507:199-220.

Unbiased Interrogation of 3D Genome Topology Using Chromosome Conformation Capture Coupled to High-Throughput Sequencing (4C-Seq).

Author information

1
Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands.
2
INSERM UMR967, CEA/DRF/iRCM, Université Paris-Diderot, Université Paris-Saclay, Fontenay-aux-Roses, France. eric.soler@inserm.fr.
3
Laboratory of Excellence GR-Ex, 75015, Paris, France. eric.soler@inserm.fr.
4
Institute of Molecular Genetics (IGMM), Laboratory of Molecular Hematopoiesis, Montpellier, 34293, France. eric.soler@inserm.fr.
5
Gene Regulation, Stem Cells andCancer Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain. Ralph.Stadhouders@crg.eu.

Abstract

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

KEYWORDS:

4C-Seq; Bioinformatics; Chromatin looping; Chromosome conformation capture (3C); DNA; Genome-wide; Long-range gene regulation

PMID:
27832542
DOI:
10.1007/978-1-4939-6518-2_15
[Indexed for MEDLINE]

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